The long term goals of our research are to learn about the mechanism for HIV-1 target recognition, to understand the selectivity of the virus to different cells of the immune system and to study mechanisms of viral entry inhibition. The binding of human immunodeficiency-virus type-1 (HIV-1) to its target cells is mediated primarily by the envelope glycoprotein (gp120) of the virus. Initially gp120 binds to CD4, a molecule found on the surface of both T-cells and macrophages. This binding triggers a conformational change in gp120 that forms another binding site to either CCR5 or CXCR4 chemokine receptors, which serve as co-receptors for HIV-1 binding. After binding to the co-receptor, gp120 undergoes another conformational change that exposes the gp41 trans-membrane glycoprotein of the virus which mediates HIV- 1 fusion with its target cell membrane. The peptide T-20 (Fuzeon), used as a viral entry inhibitor for the treatment of HIV-1 infected individuals, corresponds in its sequence to the C-terminal half of the gp41 region HR2 and a segment down stream of HR2. The structure of the co-receptor binding site on gp120 is still elusive and the mechanism for co-receptor selectivity is still unknown. Similarly, for T-20, the mechanism by which it inhibits fusion is not understood. Particularly enigmatic is the role played by its nine C-terminal residues, without which this anti-HIV drug loses its potency in entry-inhibition. The specific aims of the current proposal are as follows: a) Learn how the flexibility of gp120 enables the formation of the binding site for CD4 and CCR5, b) Learn how HIV-1 co-receptor selectivity is determined, and c) Investigate the interactions of T-20 with possible targets on gp41 and gp120 and reveal the role of the C-terminal segment T-20 in viral inhibition. We will use a novel multi-dimensional NMR technique designed to solve the structure of large proteins without requiring deuteration. The solution structure of HIV-1 gp120 in the unliganded form, in complex with a CD4-mimic peptide (miCD4) and in ternary complex with miCD4 and CCR5 extra-cellular fragments will be determined. In addition, we will determine the structure of the anti-HIV-1 drug T-20 in complex with gp41 and gp120 targets. When solved, the solution structure of HIV-1 gp120 will be probably the first structure of a protein expressed and labeled in mammalian cells and one of the largest structures solved by NMR. Considering the flexibility of gp120 that is directly related to its function, structure determination in solution is of utmost importance. The structure of gp120 in its three different conformations will enable us to learn about the mechanism for CD4 and co-receptor recognition and for designing anti-HIV- 1 immunogens, antagonists and entry inhibitors that target the gp120 and CCR5 binding sites. Studies of T- 20 complexes with gp41 and gp120 segments will reveal the role of the C-terminal segment of T-20 in viral inhibition and contribute to the design more potent entry inhibitors.